This invention relates to matrices for use in affinity chromatography and in the immobilization of biologically active materials. More specifically, this invention relates to affinity supports based on a unique series of hydrated polyurea-polyurethane polymers which have been activated to provide a means for immobilizing and chromatographing a wide variety of bioaffinity agents.
Bioaffinity separations generally involve at least one biomacromolecule, such as a protein or nucleic acid, as one of the components of the binding pair. Examples of such bioaffinity binding pairs include: antigen-antibody, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding protein-vitamin, binding protein-nucleic acid; reactive dye-protein, reactive dye-nucleic acid; and others. The terms ligand and binder will be used to represent the two bioaffinity agents in specific binding pairs. By "ligand" is meant an antigen, hapten, nucleic acid, vitamin, dye or small organic molecule including enzyme substrates, effectors, and inhibitors and the like. By binder is meant an antibody, enzyme, nucleic acid, binding protein, synthetic mimics of binding proteins such as polylysine and polyethyleneimines or other macromolecules capable of specific binding, enzyme/substrate interactions, etc. . . .
The affinity supports of the invention are based on activated polymers prepared from high molecular weight isocyanate end-capped prepolymers which are substantially comprised of ethylene oxide units. Activation of the polymers is accomplished by first derivatizing the prepolymer with a reactive compound having an NCO-reactive group and a second functional group. The derivatized or modified prepolymers are polymerized with water to yield a modified polyurea-urethane polymer characterized by the second functional group inserted into the prepolymer. The modified polyurea-urethane polymer is then activated by contacting the polymer with an activating compound to convert said second functional group to an active species which is capable of selectively covalently bonding a specific ligand or binder of interest.
Numerous polyurethane polymers have been previously identified, among them both foamed and nonfoamed materials. Of the nonfoamed materials, quite a few hydrogel polymers, prepared from various prepolymers, have been prepared and used for widely varying applications. Typically, hydrogels are formed by polymerizing a hydrophilic monomer in an aqueous solution under conditions such that the prepolymer becomes crosslinked, forming a three-dimensional polymeric network which gels the solution. Polyurethane hydrogels are formed by polymerization of isocyanate-end capped prepolymers to create urea and urethane linkages.
Representative examples of previously disclosed polyurethane hydrogels include the following: U.S. Pat. No. 4,241,537 (Wood) discloses a plant growth media comprising a hydrophilic polyurethane gel composition prepared from chain-extended polyols; random copolymerization is preferred with up to 50% propylene oxide units so that the prepolymer will be a liquid at room temperature. U.S. Pat. No. 3,939,123 (Matthews) discloses lightly crosslinked polyurethane polymers of isocyanate terminated prepolymers comprised of poly(ethyleneoxy) glycols with up to 35% of a poly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producing the Matthews polymer, an organic polyamine is used as a crosslinking agent. The Matthews prepolymers form a cross-linked, three dimensional structure when polymerized as taught in the patent. U.S. Pat. No. 4,182,827 (Jones) discloses a similar use of polyamines in the formation of polyurethane hydrogels.
Several types of compounds have been reacted with prepolymers or with matrix bases to act as spacing or coupling compounds in the attachment or immobilization of biologically active agents. For example, U.S. Pat. No. 4,226,935 (Fusee) discloses reacting an amino acid and/or a protein with an excess of a urethane prepolymer, curing the resulting product to form a polymer matrix, and coupling an enzyme thereto by use of a carbodiimide. U.S. Pat. No. 4,177,038 (Biebricher et al.) teaches the use of spacers which may be diamines, amino-alcohols or diols.
Modified polyurethane polymers also have been prepared. U.S. Pat. No. 4,439,585 (Gould et al.) teaches a polyurethane diacrylate composition obtained by reacting a diacrylate in the presence of a hydrophilic polyurethane resin. U.S. Pat. No. 4,485,227 (Fox) discloses a poly-(ether-urethane-urea) prepared by condensations of a prepolymer with primary diamines, then with an amine-reacting agent. U.S. Pat. No. 4,569,981 (Wenzel et al.) discloses water-dispersible plastics precursors based on isocyanate-terminated prepolymers which have been hydrophilically modified with ionic groups and/or ethylene oxide groups.
Biocompatibility is an increasingly desirable characteristic for polymeric hydrogels and hydrated polymers, which would find numerous uses in the health care field if the appropriate properties can be obtained. However, many conventional hydrogels and polymers are not taught to be biocompatible. In addition, modification of a polymer surface frequently results in increased nonspecific binding of unwanted proteins. Because affinity separation is a powerful technique used in laboratory and health care to purify various biologically active materials, there exists a need for affinity matrices which have improved biocompatibility and increased resistance to nonspecific protein adsorption.